Safe and Efficient Engraftment of CRISPR-Based ELANE Mono-Allelic Knocked out HSCs in Mice: Evidence for a Novel Treatment for ELANE Neutropenia

Abstract ELANE-related severe congenital neutropenia (SCN) is a disorder wherein numerous heterozygous mutations in the ELANE gene lead to misfolding and mislocalization of mutant neutrophil elastase, resulting in the death of myeloid cells and block in neutrophil differentiation. Currently, SCN is treated with daily injections of granulocyte colony-stimulating factor (G-CSF), but patients on this therapy are at risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The only other effective therapy is allogeneic hematopoietic stem cell transplantation that may involve graft versus host disease and risk of serious infections. Thus, there is great interest in safe and efficient therapeutic alternatives. Emendo Biotherapeutics has developed a novel CRISPR-based (OMNI A1) ex-vivo gene editing strategy that involves specific excision of disease-causing ELANE mutant allele. This treatment was tested on CD34 + HSCs from SCN patients and resulted in a significant improvement in the maturation of myeloid cells and their differentiation to normal functional neutrophils. To assess the feasibility and safety of this editing strategy, we conducted an in vivo study using human HSCs derived from two healthy donors that were engrafted into a NOD scid gamma (NSG) immunocompromised mice model. The study involved a single iv injection of HSCs that were either not-treated (NT), electroporated without delivery of CRISPR nuclease (mock) or edited with Emendo's novel nuclease, OMNI A1,(excision). Each group consisted of 6 NSG female mice and an additional group of 4 mice served as vehicle control (no HSCs). All animals were subjected to total body irradiation, 1 day prior to the treatment, at a dose of 250 cGy. Animals were subjected to retro-orbital blood collection, at 4 designated time points during the study (Figure A), for CD45 + cell counts using FACS. All animals were evaluated for systemic reactions, clinical signs and body weight changes during the study period. At the end of the study period, animals were subjected to peripheral blood (PB), bone marrow (BM) and spleen sampling. FACS analysis of CD45 + cells was performed on those samples to determine the engraftment efficiency. Furthermore, excision levels for ELANE were measured in the engrafted cells by droplet digital PCR (ddPCR). Finally, multilineage differentiation capacity of the engrafted cells was evaluated by FACS. Analysis of human CD45 + cells, at 20 weeks, showed comparable levels of engraftment of the not-treated, mock treated and excised HSCs in the mouse PB, BM and spleen (No statistically significant difference, Kruskal Wallis test) (Figure B). Excised cells were detected in the mouse BM and excision levels in some of the mice were comparable to those measured in the HSCs prior to engraftment (about 30-36%). Human-derived cells from the BM and spleen of mice engrafted with excised HSCs showed similar multilineage differentiation capacity as obtained in mice engrafted with either NT or mock-treated HSCs. Engrafted human CD45 + cells differentiated into neutrophils (CD66b +), myeloid cells (CD33 +), Lymphocytes: B-cells (CD19 +) and T-cells (CD3 +). In addition, a population of engrafted hematopoietic CD34 + cells was detected in the BM. Excised HSCs of both donors gave rise to all lineages tested, as efficient as the NT and the mock groups. Finally, engrafted mice showed no systemic reactions, abnormal clinical signs or loss of body weight. These data show that edited cells can be engrafted successfully, maintain their excision profile and populate the bone marrow. This study also supports the safety of the OMNI A1 therapeutic composition. Figure 1 Figure 1. Disclosures Povodovski: Emendo Biotherapeutic: Current Employment. Makaryan: Emendo Biotherapeutic: Research Funding. Sabo: Emendo Biotherapeutic: Research Funding. Dicken: Emendo Biotherapeutic: Ended employment in the past 24 months. Herman: Emendo Biotherapeutic: Current Employment. Emmanuel: Emendo Biotherapeutic: Current Employment. Dale: X4 Pharmaceuticals: Consultancy, Honoraria, Research Funding.

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The CXCL1 Inhibitor Reparixin Rescues Myelofibrosis in the Gata1low Model of the Disease

Blood ◽

10.1182/blood-2021-147261 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3579-3579

Author(s):

Paola Verachi ◽

Fabrizio Martelli ◽

Maria Zingariello ◽

Francesca Gobbo ◽

Giuseppe Sarli ◽

...

Keyword(s):

Bone Marrow ◽

Body Weight ◽

Plasma Levels ◽

Research Funding ◽

Treated Group ◽

Interleukin 8 ◽

Bone Marrow Fibrosis ◽

Advisory Committees ◽

Cell Counts ◽

Males And Females

Abstract A mayor pathobiological role for interleukin 8 in the etiology of myelofibrosis has been suggested by observations indicating that megakaryocytes expanded in culture from these patients express great levels of interleukin 8 1 and that the plasma levels of this cytokine are predictive of poor prognosis 2. In preliminary experiments we demonstrated that the megakaryocytes from the bone marrow of the Gata1 low model of myelofibrosis express not only high levels of TGF-β, but also levels greater than normal of lipokalin-2, a known inducer of IL-8 production, and of CXCL1, the murine equivalent of IL-8. In addition, these megakaryocytes express also high levels of the CXCL1 receptors CXCLR1 and CXCR2 and the bone marrow from these mice express an CXCR1/CXCR2 activated signature. Using these data as a foundation, we tested here the effects of treatment of Gata1 low mice with the CXCR1/R2 inhibitor reparixin on the myelofibrosis phenotype expressed by this models. To these aim, Gata1 low mice (8-month old) were treated either with vehicle (3 males and 3 females) or with reparixin (formerly referred to as repertaxin) 3 (5 males and 5 females) for either 20 or 37 days. The drug was administered by minipumps implanted subcutaneously in the dorsal region set to deliver 7.5mg of drug/hr/Kg of body weight. The mice receiving the drug for 37 days had the minipumps replaced by day 17. The efficiency of drug delivery decreased over time since the plasma levels of reparixin were 13.90±4.18 and 6.71±4.18ug/mL at day 20 and 37, respectively (p<0.05).The drug was well tolerated with no death or change in body weight recorded over the period of observation. Since the results observed in males and females were similar, the data were pooled for statistical analyses. The treatment did not affect blood values (hematocrit (%): 34.32±3.87 vs 35.63±3.45 and 30.92±3.58, platelets: (x10 3/uL) 187.80±26.12 vs 181.30±53.30 and 99.83±71.92 and white cell counts (x10 3/uL): 2.78±0.55 vs 3.27±0.72 and 3.57±1.43, respectively, in vehicle and day 20- or day 37-reparixin treated mice). The treatment had also little effects on bone marrow (20.55±5.83 vs 22.24±0.85 and 21.68±6.49) and on spleen 141.40±29.04 vs 99.54±15.55 and 173.00±76.54) cellularity. However, the bones were reddish and their sections contained great numbers of erythroid cells, a sign of increased hematopoiesis. Great reductions in the fibrosis of the bone marrow and spleen was observed in mice that had been treated with reparixin compared to vehicle which were statistically significant by day 20 (day 20 bone marrow fibrosis 28.09±15.69 in vehicle and 4.54±0.45 in reparixin treated mice by Gomori, p<0.05; 19.30±7.86 vs 3.19±1.89 by reticulin, staining, p<0.05, respectively by Anova; day 20 spleen fibrosis 20.51±5.25 in vehicle and 10.85±3.82 in reparixin treated mice by Gomori, p<0.05; and 13.15±3.06 vs 6.13±2.34 by reticulin, staining, p<0.05, respectively). Of note when the levels of Gomori and reticulin fibrosis detected at day 20 and 37 in individual mice were inversely correlated with the plasma levels of reparixin observed in the same mice (Figure 1, p<0.01-0.05 by Pearson). Mechanistic insights on these results were provided by Immunostaining of marrow and spleen sections of vehicle and reparixin-treated mice indicating that the megakaryocytes from the reparixin-treated group express levels of TGF-β significantly lower than those expressed by the corresponding cells from vehicle while the levels of LCN-2, CXCL1, CXCR1 and CXCR2 expressed by the reparixin treated megakaryocytes are similar to that of the vehicle treated cells. These results indicate that inhibition of CXCL1 by reparixin, probably by reducing the abnormally high TGF-β content of the megakaryocytes, reduces fibrosis in Gata1 low mice and provide a preclinical rational to test this drug in patients with myelofibrosis. References: 1) Emadi S et al. Blood. 2005;105:464; 2) Tefferi et al, J Clin Oncol. 2011;29:1356; 3) Bertini R et al, PNAS 2004; 101:11791 Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Massucci: Dompe Farmaceutici Spa R&D: Current Employment. Brandolini: Dompe farmaceutici Spa R&D: Current Employment. Giorgio: Dompe farmaceutici Spa R&D: Current Employment. Allegretti: Dompe farmaceutici Spa R&D: Current Employment. Migliaccio: Dompe farmaceutici Spa R&D: Other: received funding for reserach .

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Dnmt3a Is Essential for Hematopoietic Stem Cell Differentiation

Blood ◽

10.1182/blood.v118.21.386.386 ◽

2011 ◽

Vol 118 (21) ◽

pp. 386-386 ◽

Cited By ~ 1

Author(s):

Grant A. Challen ◽

Deqiang Sun ◽

Mira Jeong ◽

Min Luo ◽

Jaroslav Jelinek ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Dna Methyltransferase ◽

Donor Cell ◽

The Self ◽

Blood Cell Count ◽

Serial Transfer ◽

Self Renewal ◽

Hematopoietic Stem ◽

Differentiation Capacity

Abstract Abstract 386 Aberrant genomic DNA methylation patterns are widely reported in human cancers but the prognostic value and pathological consequences of these marks remain uncertain. CpG methylation is catalyzed by a family of DNA methyltransferase enzymes comprised of three members – Dnmt1, Dnmt3a and Dnmt3b. Mutations in the de novo DNA methyltransferase enzyme DNMT3A have now been reported in over 20% of adult acute myeloid leukemia (AML) and 10–15% of myelodysplastic syndrome (MDS) patients. However, analysis of promoter methylation and gene expression in these patients has thus far failed to yield any mechanistic insight into the pathology of DNMT3A mutation-driven leukemia. In this study, we have used a conditional knockout mouse model to study the role of Dnmt3a in normal hematopoiesis. Hematopoietic stem cells (HSCs) from Mx1-Cre:Dnmt3afl/fl mice were serially transplanted into lethally irradiated recipient mice to study the effect of loss of Dnmt3a on HSC self-renewal and differentiation. We show that loss of Dnmt3a progressively impedes HSC differentiation over four-rounds of serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Examination of the bone marrow post-transplant revealed that control HSCs showed a gradual decline in their ability to regenerate the HSC pool at each successive round of transplantation, while in contrast Dnmt3a-KO HSCs show a remarkably robust capacity for amplification, generating 40,000 – 100,000 HSCs per mouse. Quantification of peripheral blood differentiation on a per HSC basis demonstrated in the absence of Dnmt3a, a cell division is more likely to result in a self-renewal rather than differentiation fate (Figure 1). Using semi-global reduced representation bisulfite sequencing (RRBS), we show that Dnmt3a-KO HSCs manifest both increased and decreased methylation at distinct loci, including dramatic CpG island hypermethylation. Global transcriptional analysis by microarray revealed that Dnmt3a-KO HSCs show upregulation of HSC multipotency genes coupled with simultaneous downregulation of early differentiation factors (e.g. Flt3, PU.1, Mef2c), likely inhibiting the initial stages of HSC differentiation. Upregulation of key HSC regulators including Runx1, Gata3 and Nr4a2 was associated with gene-body hypomethylation and activated chromatin marks (H3K4me3) in Dnmt3a-KO HSCs. Finally, we show that Dnmt3a-KO HSCs are unable to methylate and transcriptionally repress these key HSC multipotency genes in response to chemotherapeutic ablation of the hematopoietic system, leading to inefficient differentiation and manifesting hypomethylation and incomplete repression of HSC-specific genes in their limited differentiated progeny. In conclusion, we show that Dnmt3a plays a specific role in permitting HSC differentiation, as in its absence, phenotypically normal but impotent stem cells accumulate and differentiation capacity is progressively lost. This differentiation-deficit phenotype is reminiscent of Dnmt3a/Dnmt3b-null embryonic stem (ES) cells while markedly distinct from that of Dnmt1-KO HSCs which show premature HSC exhaustion and lymphoid-deficient differentiation, demonstrating distinct roles for the different DNA methyltransferase enzymes in HSCs. In light of the recently-identified DNMT3A mutations in AML and MDS patients, these studies are the first biological models linking mutation of Dnmt3a with inhibition of HSC differentiation which may be one of the first pathogenic steps occuring in such patients.Figure 1Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal.Figure 1. Dnmt3a-KO HSCs become biased towards self-renewal as opposed to differentiation. At each transplant round, the self-renewal quotient was calculated as the number of donor-derived HSCs recovered at the end of the transplant divided by 250 (the number of HSC initially transplanted). The differentiation quotient was calculated as (the white blood cell count per μl of blood at 16 weeks) X (percentage of donor-cell chimerism)/number of donor HSC at the end of the transplant. Over serial transfer, Dnmt3a-KO HSCs more rapidly lose their differentiation capacity compared to control HSCs, while sustaining robust self-renewal. Disclosures: Issa: Novartis: Honoraria; GSK: Consultancy; SYNDAX: Consultancy; Merck: Research Funding; Eisai: Research Funding; Celgene: Research Funding; Celgene: Honoraria; J&J: Honoraria.

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THROMBOCYTOSIS: A RETROSPECTIVE STUDY OF 573 DOGS (2016-2017)

Ciência Animal Brasileira ◽

10.1590/1089-6891v20e-51837 ◽

2019 ◽

Vol 20 ◽

Author(s):

Marcela Natacha Aparecida Rocha ◽

Mayara Carvalho de Sousa Rocha ◽

Mayara Lima Kavasaki ◽

Juliana Yuki Rodrigues ◽

Weyber Ferreira de Souza ◽

...

Keyword(s):

Clinical Signs ◽

Clinical Importance ◽

Blood Platelet ◽

Gastrointestinal Diseases ◽

Platelet Counts ◽

Cell Counts ◽

Significant Difference ◽

Underlying Causes ◽

Clinical Conditions ◽

Degree Of Severity

Abstract Thrombocytosis refers to the increase in number of platelets per microliter (µL) of blood. Platelet counts greater than 1,000,000/µL may be associated with clinical signs of bleeding or thrombosis. Previous studies on underlying causes of thrombocytosis have aroused the interest of researchers about its clinical importance in dogs. The objective of this study was to analyze the blood cell counts in dogs in order to define the main diseases or clinical conditions that were associated with thrombocytosis, from 2016 to 2017. This was done to determine the incidence of thrombocytosis, and categorize the increase in platelet count with respect to severity. Of the 12,676 blood samples analyzed, 4.5% presented thrombocytosis (n = 573). Similar mean platelet counts were observed in all diagnosis or different categories of clinical conditions (neoplasms; gastrointestinal, endocrine, and ophthalmological diseases; trauma and surgery; dermatological, cardiac, neurological, infectious, respiratory, genitourinary, idiopathic, and multiple diseases; and pregnancy) with no significant difference (P ≥ 0.05). The disorders most commonly associated with thrombocytosis were gastrointestinal diseases, followed by neoplasms. Furthermore, increased platelet counts were observed in dogs treated with glucocorticoids and vincristine drugs. As for the degree of severity, extreme thrombocytosis occurred more frequently in the presence of gastrointestinal diseases.

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Changes in Bone Marrow Fat upon Dietary-Induced Weight Loss

Nutrients ◽

10.3390/nu12051509 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1509

Author(s):

Manuela Spurny ◽

Yixin Jiang ◽

Solomon A. Sowah ◽

Ruth Schübel ◽

Tobias Nonnenmacher ◽

...

Keyword(s):

Bone Marrow ◽

Weight Loss ◽

Study Cohort ◽

Marrow Fat ◽

Bone Marrow Fat ◽

Randomized Controlled ◽

Cell Counts ◽

Significant Difference ◽

Metabolic Biomarkers ◽

The Impact

Background: Bone marrow fat is implicated in metabolism, bone health and haematological diseases. Thus, this study aims to analyse the impact of moderate weight loss on bone marrow fat content (BMFC) in obese, healthy individuals. Methods: Data of the HELENA-Trial (Healthy nutrition and energy restriction as cancer prevention strategies: a randomized controlled intervention trial), a randomized controlled trial (RCT) among 137 non-smoking, overweight or obese participants, were analysed to quantify the Magnetic Resonance Imaging (MRI)-derived BMFC at baseline, after a 12-week dietary intervention phase, and after a 50-week follow-up. The study cohort was classified into quartiles based on changes in body weight between baseline and week 12. Changes in BMFC in respect of weight loss were analysed by linear mixed models. Spearman’s coefficients were used to assess correlations between anthropometric parameters, blood biochemical markers, blood cells and BMFC. Results: Relative changes in BMFC from baseline to week 12 were 0.0 ± 0.2%, −3.2 ± 0.1%, −6.1 ± 0.2% and −11.5 ± 0.6% for Q1 to Q4. Across all four quartiles and for the two-group comparison, Q1 versus Q4, there was a significant difference (p < 0.05) for changes in BMFC. BMFC was not associated with blood cell counts and showed only weaker correlations (<0.3) with metabolic biomarkers. Conclusion: Weight loss is associated with a decrease of BMFC. However, BMFC showed no stronger associations with inflammatory and metabolic biomarkers.

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Granulocyte colony-stimulating factor crosses the placenta and stimulates fetal rat granulopoiesis

Blood ◽

10.1182/blood.v81.4.916.916 ◽

1993 ◽

Vol 81 (4) ◽

pp. 916-922 ◽

Cited By ~ 57

Author(s):

ES Medlock ◽

DL Kaplan ◽

M Cecchini ◽

TR Ulich ◽

J del Castillo ◽

...

Keyword(s):

Bone Marrow ◽

Myeloid Cells ◽

Serum Levels ◽

Polymorphonuclear Cells ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Pregnant Rats ◽

Cell Counts ◽

Fetal Serum ◽

Stimulating Factor

Abstract We studied the effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) administration to pregnant rats upon fetal and neonatal myelopoiesis. Pregnant rats were treated with rhG-CSF twice daily for 2, 4, and 6 days before parturition. rhG-CSF crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. Peak fetal serum levels were 1,000-fold lower than levels detected in the dam. Hematopoietic effects of rhG-CSF were assessed by cytologic analysis of the newborn blood, spleen, bone marrow, thymus, and liver. White blood cell counts were increased twofold to fourfold in newborns. This increase was due to circulating numbers of polymorphonuclear cells (PMN). rhG-CSF induced a myeloid hyperplasia in the newborn marrow consisting of immature and mature myeloid cells in the day-2 and day-4 treated pups. Bone marrow of pups treated for 6 days contained mostly hyper-segmented PMN with little or no increase in myeloid precursors. An increase in the number of postmitotic (PMN, bands, and metamyelocytes) and mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid cells in the spleen of neonates was observed. No change was detected in splenic lymphocytes or monocytes. No effect of rhG-CSF was noted in the newborn liver or thymus. These results demonstrate that maternally administered rhG-CSF crosses the placenta and specifically induces bone marrow and spleen myelopoiesis in the fetus and neonate. The significant myelopoietic effects of rhG-CSF at low concentrations in the fetus suggest an exquisite degree of developmental sensitivity to this cytokine and may provide enhanced defense mechanisms to the neonate.

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The Auto-Antibodies on Bone Marrow Cells in the Patients with Systemic Lupus Erythematosus.

Blood ◽

10.1182/blood.v108.11.3856.3856 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3856-3856

Author(s):

Rong Fu ◽

Jizi Deng ◽

Shang Yuan ◽

Lu Gong ◽

Jun Sun ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Bone Marrow ◽

Lupus Erythematosus ◽

Mononuclear Cells ◽

Healthy Controls ◽

Coombs Test ◽

Systemic Lupus ◽

Cell Counts ◽

Significant Difference ◽

Positive Rate

Abstract Objective:To explore the pathogenesis of cytopenia in the patients with systemic lupus erythematosus (SLE), the auto-antibodies on bone marrow mononuclear cells (BMMBC) in the patients with SLE were determined. Methods:Twenty one patients with SLE and ten healthy controls were enrolled in this study. BMMNC Coombs test was used to determine the aotoantibodies. The correlation between the types of auto-antibodies on BMMNC, the types of serum auto-antibodies and the counts of blood cells in the patients with SLE were also investigated. Results:Positive results of BMMBC-Coombs test were seen in 12 patients with SLE (57.1%), among them, 10 with hemocytopenia (58.82%), and 2 without hemocytopenia (50%). The positive rate of BMMNC Coombs test was higher in the patients with SLE than that in healthy controls, and was higher in SLE patients with hemocytopenia than that in healthy controls. There were no significant difference of BMMNC-Coombs positive rate between the SLE patients without hemocytopenia and healthy controls, and there were also no significant differences between the SLE patients without hemocytopenia and SLE patients with hemocytopenia. In the 12 SLE patients with positive BMMBC-Coombs tests, IgM auto-antibody accounted for 75.0%, and C3 50.0%, IgG 8.33%, IgG+IgM 8.33%, C3+IgM 16.67%, IgG+IgM+C3 16.67%. In the SLE patients without hemocytopenia, IgG+IgM accounted for 8.33%, C3 8.33%, but IgA autoantibody were not seen in any case. There was a significant positive correlation between the auto-antibodies on BMMNC and peripheral anti-SSA, but there was no significant correlation between the results of BMMBC Coombs tests and peripheral blood cell counts. Conclusion:There were auto-antibodies on BMMNC in the patients with SLE. The hemocytopenia in the patients with SLE maybe resulted from the destructions of bone marrow hematopoietic cells by the autoantibodies.

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Evidence for An Anti-Cancer Barrier in a Mixed Lineage Leukemia Mouse Model in Vivo.

Blood ◽

10.1182/blood.v112.11.3347.3347 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3347-3347

Author(s):

Sylvia Takacova ◽

Jiri Bartek ◽

Lucie Piterkova ◽

Robert K. Slany ◽

Vladimir Divoky

Keyword(s):

Bone Marrow ◽

Tumor Suppressor ◽

Mouse Model ◽

Peripheral Blood ◽

Myeloid Cells ◽

Mixed Lineage Leukemia ◽

Cell Counts ◽

Potential Tumor Suppressor

Abstract Mixed Lineage Leukemia (MLL) mutations identify a unique group of acute leukemias with distinct biological and clinical features. Although the role of MLL in leukemogenesis has been extensively studied, a precise mechanism regarding the leukemogenic potential of MLL mutations is not known. We generated a switchable MLL-ENL-ERtm mouse model, in which the MLL-ENL oncogene has been introduced by homologous recombination and is controlled by the endogenous MLL promoter, thus, expressed at physiological levels. Due to fusion with the estrogen receptor ligand binding domain (ERtm), the MLL-ENL-ERtm protein activity is dependent on continuous provision of tamoxifen or 4-hydroxytamoxifen. The MLL-ENL-ERtm mice have developed a myeloproliferative disorder (MPD) characterized by persistent mature neutrophilia after 484,5 +/− 75,68 days of latency on a tamoxifen diet, in association with high white cell counts in peripheral blood, splenomegaly and occasionally with anemia. Blood smears showed large numbers of mature myeloid elements consisting of 40–80% neutrophils (non-segmented forms in abundance), admixed with immature myeloid elements, 3–11% monocytes and 2–6% myeloblasts. The phenotype of MPD also involved myelomonocytic proliferation with 35% immature monocytic cells in one animal and severe anemia with increased numbers of immature erythroid cells in peripheral blood in another animal. Hematoxylin- and eosin-stained sections of the bone marrow from MLL-ENL-ERtm mice revealed expansion of myeloid cell population with no signs of progressive dysplasia. We observed massive infiltration of myeloid cells (positive for myeloperoxidase) into spleen with various degree of loss of normal splenic architecture depending on disease progression. FACS profiles of both bone marrow and spleen cells showed a typical pattern of granulocyte/macrophage/monocyte surface marker expression (CD34-CD43+Mac- 1+Gr-1+CD16/32+). In vitro evaluation of hematopoetic progenitors derived from bone marrow of leukemic mice at the terminal stage of the disease revealed decreased numbers of BFU-Es and increased numbers of CFU-GMs and CFU-Gs compared to matched controls. These results correlated with the expansion of the myelomonocytic and reduction of the erythroid compartment observed in the bone marrow of these animals. The average size (cellularity) of the mutant myeloid colonies was much smaller than the colonies derived from the wild-type controls, which could be caused by a partial block of terminal differentiation of myeloid progenitors in vitro. In vivo, MLL-ENL leads to expansion of differentiated myeloid cells in our model. High penetrance and long latency of leukemia in our model permits the study of early leukemia development. Our model revealed that MLL-ENL - induced myeloproliferation occurs as early as twelve weeks after MLL-ENL-ERtm activation in the bone marrow and infiltrates the spleen with a consequent decrease in lymphoid B220+CD19+IgM+ cells. Using the TUNEL assay on bone marrow sections, we observed induction of apoptosis in the highly proliferative bone marrow compartment compared to matched controls. These results suggest activation of a potential tumor suppressor mechanism by MLL-ENL in early stages of leukemia. We are currently investigating potential tumor suppressor pathways that might be involved in MLL-ENL - induced apoptosis in preleukemia.

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Gabp Transcription Factor Is Required for Self-Renew and Proliferation of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v118.21.1284.1284 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1284-1284

Author(s):

Zhongfa Yang ◽

Karen Drumea ◽

James Cormier ◽

Junling Wang ◽

Xuejun Zhu ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Transcription Factor ◽

Progenitor Cells ◽

Myeloid Cells ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Significant Difference ◽

Stem And Progenitor Cells ◽

Ets Domain

Abstract Abstract 1284 GABP is an ets transcription factor that regulates genes which are required for normal hematopoietic development. In myeloid cells, GABP is an essential component of a retinoic acid-inducible enhanceosome that mediates granulocytic gene expression and, in lymphoid cells, GABP regulates expression of IL7-R and the essential transcription factor, Pax5. GABP is a tetrameric complex that includes GABPa, which binds DNA via its ets domain, and GABPb, which contains the transcription activation domain. Genetic disruption of mouse Gabpa caused early embryonic lethality. We created mice in which loxP recombination sites flank exons that encode the Gabpa ets domain, and bred them to mice that bear the Mx1Cre recombinase; injection with pIC induced Cre expression and efficiently deleted Gabpa in hematopoietic cells. One half of the Gabpa knock-out (KO) mice died within two weeks of pIC injection in association with widespread visceral hemorrhage. Gabpa KO mice exhibited a rapid loss of mature granulocytes, and residual myeloid cells exhibited myelodysplasia due, in part, to regulation by Gabp of the transcriptional repressor, Gfi-1. We used bone marrow transplantation to demonstrate that the defect in Gabpa null myeloid cells is cell intrinsic. Although hematopoietic progenitor cells in Gabpa KO bone marrow were decreased more than 100-fold compared to pIC treated control mice, there was not a statistically significant difference in the numbers of Lin−c-kit+Sca-1− hematopoietic stem cells (HSCs) between KO and control mice. Genetic disruption of Gfi-1 disruption in HSCs caused increased cell cycle activity – an effect that is diametrically opposite of the effect of Gabpa KO; this suggests that the effect of Gabpa on HSCs is not due to its control of Gfi-1. In contrast, Gabpa KO HSCs exhibited a marked decrease in cell cycle activity, but did not demonstrate increased apoptosis. The defects in S phase entry of Gabpa null HSCs are reminiscent of the cell cycle defects in Gabpa null fibroblasts, in which expression of Skp2 E3 ubiquitin ligase, which controls degradation of the cyclin dependent kinase inhibitors (CDKIs) p21 and p27, was markedly reduced following Gabpa disruption. We showed that Gabpa KO cells express reduced levels of Skp2. We propose that GABP controls self-renewal and proliferation of mouse bone marrow stem and progenitor cells, in part, through its regulation of Skp2. Thus, Gabpa is a key regulator of myeloid differentiation through its control of Gfi-1, but it is required for cell cycle activity of HSCs, by a distinct effect that may be due to its control of Skp2 and CDKIs. Disclosures: No relevant conflicts of interest to declare.

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“RUNX1-IRES-GFP Knock-in” Mice Lack Expression of Runx1a: A Versatile Tool for Hematopoietic Stem Cell Analysis

Blood ◽

10.1182/blood.v118.21.2329.2329 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2329-2329

Author(s):

Yukiko Komeno ◽

Ming Yan ◽

Shinobu Matsuura ◽

Miao-Chia Lo ◽

James R. Downing ◽

...

Keyword(s):

Body Weight ◽

Conflicts Of Interest ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Blood Indices ◽

Cell Counts ◽

Significant Difference ◽

Versatile Tool ◽

Exon 4 ◽

Runx1 Expression

Abstract Abstract 2329 Previously reported “RUNX1-IRES-GFP knock-in mice” (Blood 2004;103:2522) (KI mice) were generated by replacing exon 4 of runx1 gene with cDNA of Runx1b/c from exon 4 to exon 8 followed by IRES-GFP, aiming to evaluate Runx1 expression in specific lineages and developmental stages during adult hematopoiesis. They are phenotypically normal, fertile, and blood indices are normal. GFP intensity correlates with Runx1 expression level, and shows lineage-specific changes during maturation in myeloid, erythroid, and lymphoid cells. However, the behavior in the hematopoietic stem cells (HSCs) had not been carefully examined. Interestingly, we discovered that this knock-in strategy eliminated Runx1a expression. Since Runx1a expression is relatively higher in HSCs than in differentiated cells, we analyzed HSCs in these mice to evaluate its roles in stable and stress hematopoiesis. We found that LSK fraction in bone marrow (BM) was significantly decreased in KI mice compared to wild type (WT) mice (0.043% vs 0.085%, p = 0.001). Among subpopulations in LSK, short-term HSC and multipotent progenitor fractions were significantly decreased (0.024% vs 0.046%, p = 0.003, 0.0021% vs 0.0026%, p = 0.001, respectively). SLAM marker staining using CD150 and CD48 showed similar results. Competitive repopulation assay showed less functional HSCs in KI mice. However, there was no significant difference in recovery of cell counts after single-dose 5-FU intraperitoneal injection (150 mg/kg body weight) or sublethal irradiation (5 Gy), or survival after weekly 5-FU injection. After G-CSF subcutaneous injection (125 μg/kg body weight, twice daily for 5 days), mobilized WBC or neutrophil in PB showed no difference. However, LSK and long-term HSC in PB were significantly less in KI mice (0.078% vs 0.135%, p = 0.010, 0.043% vs 0.092%, p = 0.029, respectively) while those in BM did not show significant difference (increased to 0.295% and 0.346% in KI and WT mice, respectively). In conclusion, Runx1a plays some non-redundant roles in stable hematopoiesis, while it is dispensable for tested stress hematopoiesis. RUNX1-GFP KI mice are a versatile tool to evaluate roles of Runx1a in normal hematopoiesis and leukemogenesis when combined with other genetic modifications. Disclosures: No relevant conflicts of interest to declare.

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Fertility Recovery Following Allogeneic Bone Marrow Transplantation in Aplastic Anemia; A Study of 157 Patients

Blood ◽

10.1182/blood.v126.23.4783.4783 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4783-4783

Author(s):

Feras Alfraih ◽

Shad Ahmed ◽

Dennis Dong Hwan Kim ◽

Walid Rasheed ◽

Ghuzayel Aldawsari ◽

...

Keyword(s):

Risk Factors ◽

Bone Marrow ◽

Risk Factor ◽

Aplastic Anemia ◽

Median Duration ◽

Research Funding ◽

Independent Risk Factor ◽

Age Group ◽

Significant Difference ◽

Fertility Recovery

Abstract Introduction : Infertility is a major late effect of hematopoietic stem cell transplants (HSCT). In aplastic anemia (AA) patients, although the fertility recovery rate is relatively higher than other diseases but the exact incidence and risk factors are not very well studied. In this study, we attempted to evaluate incidence and the impact of patientÕs characteristics and transplantation procedures on fertility recovery following allogeneic HSCT for adolescent and adults patients with AA. Methods : A total of 157 patients who were at least 14 years old with AA receiving HSCT between year 1987 and 2014 at our center were reviewed. Patients who survived at least 2 years following HSCT and either married or in relationship were included in the analysis and evaluated for fertility following HSCT. 87 patients were eligible for the study. Questionnaire survey and long-term charts were used for data collection. With a response rate and or available information of 63% patients, 55 patients were identified and stratified into fertility recovery (FR+) versus non-fertility recovery (FR-) group. Fertility recovery was defined by a pregnancy of the patient or his partner. Results: Median age for all patients is 23 years (range, 14 -50), 44% (n=24) between 14-20 years old, 51% (n=28) between age 20-40 years and 5% (n=3) > 40 years. 51% (n=28) were females. Matched related donor was used for majority of patients 96% (n=53). GVHD prophylaxis was CSA/MTX for 93% (n=51,). Conditioning regimen was Cyclophosphamide/Flu in 25 (45%), Cyclophosphamide /ATG in 18 patients (35%) and others in 12 patients (20%). Bone marrow was the source of stem cells for 52 patients (94%). A median follow-up of 8 years for survivors (range, 0.3 -23) showed 45 patients (82%) had FR+ while 10 patients (18%) were FR-. Median duration of fertility recovery (from delivery to BMT) was 6 years (range, 0.8-19) with significant difference based on age groups, 4 years for patients 20-40 years (n=29, 53%) versus 8 years for those < 20 years (n=24, 44%), (p=0.002), (Figure 1). None of the patients >40 years old (n=2, 4%) had fertility recovery. Comparison based on gender showed no significant difference. Males had a median duration of fertility recovery of 5.9 years, (range 0.6-14.9) versus 6.2 years, (range, 0.8-15.2) (p=0.31) females. The overall median number of pregnancies was 2 (range, 1-6). For males, it was 2 (range, 1-6) while 1.5 (range, 1-5) for females (p=0.26). Deliveries occurred in natural ways in (95%) while C-section for (5%). All deliveries were without fetal abnormalities. Univariate analysis of risk factors for fertility recovery showed age group (p=0.03) and chronic GVHD (p=0.05) are important factors. Neither gender of patients or type of preparative regimens used for HSCT (Cyclo/ATG vs Cyclo/Flu) was a risk factor. In multivariate analysis, age group was the only confirmed an independent risk factor for fertility recovery (p=0.02) [HR= 2.02, CI=1.012-3.64). Conclusion: The present study suggested that the incidence of fertility recovery following HSCT for patients with aplastic anemia is high with no significant differences between males and females. Patients between the ages of 20-40 years at the time of HSCT have significantly shorter recovery period. Age was the only independent risk factor for fertility recovery while there was no impact of whether ATG or Fludarabine was used in addition to Cyclophosphamide as preparative regimen. Figure 1. Figure 1. Disclosures Kim: Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

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